Open projects at Department of Health Technology

• Link to websites: Drug Delivery and Sensing (IDUN)
• Overall research theme(s) in lab: Design, fabrication and tailoring of multilayer naofiber-based constructs for tunable drug delivery and engineering of complex tissues. Depending on the route of interest and thanks to the flexibility of the nanofibers, constructs of various forms including sheets, roles, and microparticles are developed. The aim is to encapsulate drug (anticancer compounds)-loaded nanocarriers into nanofibers for sustained delivery in local or oral applications.

• Link to websites: https://www.cancer.dk/danish-cancer-institute/research-groups/cellular-homeostasis-and-recycling/
• Overall research theme(s) in lab: The majority of projects in the group are centred around the process of autophagy, a conserved and essential cellular quality control pathway. We study how the process works at a mechanistic level, how it contributes to disease including cancer initiation and progression as well as how its manipulation can alter therapeutic outcomes. With this angle, we focus on obtaining a deeper mechanistic understanding of cancer cell biology.

• What?
  • Multilayer Nanostructured Microparticles for colon targeted delivery.
• How?
  • We will apply electrohydrodynamic techniques to fabricate multilayers (consisting of fibers and particles loaded by selected components) which will be cut to form nanostructured microparticles of the same size and shape. We will further perform in vitro, ex vivo and in vivo studies to assess the efficacy of applied APIs for the target study case of colorectal cacer treatment.
• Why?
  • Local administration of anticancer components, based on hydrogels and/or microparticles, have shown increased drug accumulation at the tumor site while decreasing systemic toxicity. Therefore, we are going to study the efficacy of our nanostructured microparticles for increased delivery to colon and enhanced the anticancer efficacy and cellulr uptake at the tumor site; either as an independent system or as accompanying the conventional chemotherapy.

Cancer treatment modalities have extended from traditional surgery, chemotherapy, and radiation therapy to new therapeutic approaches including targeted therapy. To increase the prognosis and quality of life of patients with cancer, an interdisciplinary approach especially for bridging engineering and biology is required. The first part of this project focuses on development of specific microparticles for colon targeted delivery. This part includes design, fabrication and in vitro/in vivo characterization of (multi)drug-loaded nanofibers which are cut into microparticles. Based on drug release studies in vitro and in vivo to validate target delivery to colon, we move towards the biological studies as the second part of the project. This part includes in depth in vitro (and potentially some in vivo) studies on efficacy of the anticancer treatment against selected cell lines/induced tumours. We will perform a comprehensive cellular/molecular analysis to investigate the efficacy of our technique, including analysis of downstream stress-response signalling pathways and investigation of viability, apoptosis, proliferation and migration in colon cancer cell lines. In addition, we will use a range of imaging technologies to investigate the mechanism of cellular uptake and intracellular localization of the microparticles.

Our research aims to advance the understanding of ocular diseases and develop therapies to preserve vision. We leverage stem cell-derived eye organoids, patient-specific disease models, and advanced techniques, including spinning disk confocal microscopy, single cell transcriptomics and electrophysiology. In this project we will focus on developing eye organoids to study retinal diseases such as diabetic retinopathy.

• https://bric.ku.dk/people/issazadeh_group/?pure=en/persons/326048 
• In the Issazadeh-Navikas group we focus on neuronal immunity and the interaction between the immune and central nervous systems, gene regulation and neuroinflammation. In this project we will investigate the neuroinflammatory response of retinal neurons in hyperglycemic conditions. We have vast experience in advanced methodologies to investigate immune-neuronal crosstalk and mitochondrial health, as well as techniques to assess potential immune cell involvement, including multi-Omics that will bring important insights to this project.

• What?
  • Development of a diabetic retinopathy (DR) organoid model focusing on neuro-inflamation in DR and diabetic retinal neurodegeneration (DRN).
• How?
  • We suggest to use a whole eye organoid model, first developed by Hayasy et al.¹, termed “SEAM”, where human pluripotent stem cells, defferentiate to form concentric ring-like zones of ocular ectoderm, including a neuronal zone with retinal progenitor cells (photo receptors and ganglion cells). The SEAM will be made diabetic by culturing the organoids in high glucose media. An osmotic control using D-mannitol. We will evaluate the induction of a neuro inflammatory response by quantifying the expression of imflamatory markers related to DR (IL-1, VEGF and MCP-1²), qPCR and WB. Loss of nerve-cell, morphology and possible involvment of immune-cells will be investigated using state of the art microscopy techniques.
• Why?
  • DR, a leading cause of blindness, has long been considered to be a vascular disease, but evidens suggest that neuro immflamation and neuro degredation predates vascular involvment in the disease³. The development of biologically relevant models is nessesarry to further our understanding and for the development of new treatments that traget the early stages of the disease. The SEAM organoid model has the potential to be used as such a model for DR, because it develops retinal neurons in a simi-2D organoid culture that can be cultured for extended time, with out of specific cell types in the “necrotic core” found in 3D spherical organoids. Additionally the generation of glia-like cells, akin to the recident immune cells of the retina has been reported in SEAM⁴ and offers a unique opportunity to study their involvment in this model.

1. Hayashi, R. et al. Co-ordinated ocular development from human iPS cells and recovery of corneal function. Nature 531, 376–380 (2016).
2. de Lemos, L. et al. Modelling neurodegeneration and inflammation in early diabetic retinopathy using 3D human retinal organoids. Vitr. Model. 3, 33–48 (2024).
3. Rübsam, A., Parikh, S. & Fort, P. E. Role of inflammation in diabetic retinopathy. Int. J. Mol. Sci. 19, 942 (2018).
4. Shiraki, N. et al. PAX6-positive microglia evolve locally in hiPSC-derived ocular organoids. Stem Cell Reports 17, 221–230 (2022).

This project combines stem cell biology and organoid technology with neuroinflammation research to investigate the early mechanisms underlying DR. By integrating expertise from these two distinct fields, the project will provide a comprehensive understanding of how retinal neurons and glial cells respond to diabetic conditions and how immune signaling pathways contribute to neurodegeneration in DR.
To achieve this, we will:

1. Establish a DR model using SEAM. The first task will be to develop a reliable protocol for creating a diabetic SEAM model by optimizing glucose concentrations and maturation time. The model will be validated by measuring hallmark signals associated with DR, such as increased expression of cytokines (e.g., IL-1β, VEGF, MCP-1) and activation of the mTOR pathway. This step involves engineering the model to recapitulate the inflammatory and neurodegenerative environment of DR, laying the foundation for further studies.
2. Analyzing the neuro-inflammatory response and immune cell involvement in DR. A central aspect of this project is the development of a "disease-in-a-dish" model that incorporates both retinal neurons and immune-like glial cells. Using this system, we aim to evaluate the induction of neuroinflammation by quantifying inflammatory markers, studying changes in neuronal morphology, and identifying potential immune cell involvement. Advanced imaging and molecular techniques will be used to investigate how neuronal health and immune signaling interact in the diabetic retina.
3. Bridging expertise across disciplines. The development of a SEAM-based DR model combines expertise in stem cell differentiation, organoid culture, and retinal biology. Simultaneously, the focus on neuroinflammation leverages methods and insights from immunology and CNS biology. The integration of these disciplines allows for a novel approach to studying early DR pathogenesis, emphasizing the interplay between retinal neurons, glial cells, and immune signaling pathways.

• https://www.healthtech.dtu.dk/research/research-sections/section-xti/group-t-cell-antigens-and-immunogenicity
• Our overall aim is to discover novel T-cell targets (antigens) and understand the adaptation of T-cells in different disease settings. We perform extensive evaluations of T-cells using cutting-edge technologies that we have established over the years. Identification of new T-cell targets and their details characterization in cancer, viral infections, and vaccination is key to improving ongoing T-cell therapies and identifying new avenues for therapeutic application

Clinical Professor Kirsten Grønbæk

• https://www.bric.ku.dk/research-groups/Research/groenbaek-group/

• Focuses on improving the survival of patients with blood cancers. To better understand the complexity of hematological cancer, the Grønbæk Group seeks an in-depth understanding of the molecular background of these diseases. In the Grønbæk group, we specifically investigate the role and interaction between genetics and epigenetics in the origin and evolution of hematological cancer.

Professor Fran Supek

• https://www.bric.ku.dk/research-groups/Research/supek-group/
• Has expertise in studying genomic and structural variants using computational analysis and machine learning approaches. Performs large-scale bioinformatic studies of multi-omic data from human tumors (somatic mutations, epigenomes, and transcriptomes), human populations (germline variation), and metagenomes (incl. human microbiomes). 

Mutations in splicing factors (e.g., SF3B1, SRSF2, U2AF1) are widespread in hematological cancers. These mutations can lead to prevalent production of defective or altered proteins due to error-prone mRNA splicing, and could potentially create a large pool of T-cell antigens. In this project, we aim to identify and evaluate the impact of CD8+ T-cells reactive to antigens derived from alternative splicing in MDS (Myelodysplastic syndromes) and CLL (chronic lymphocytic leukemia) patients.

Based on the genomic profiling of MDS and CLL patients, patients with mutations in splicing factors will be identified and a computational pipeline will be established to identify cancer-specific transcripts. A large set of candidate antigens identified in this process will be evaluated for their T-cell reactivity using our state-of-the-art DNA-barcode-based T-cell detection platform in corresponding patient samples. Furthermore, we will perform a comprehensive characterization of TCR-specificity, transcriptomics, and phenotype of splice-antigen-specific T-cells using single-cell analysis in treatment naïve and patients treated with immune checkpoint inhibitors. This project goes beyond conventional mutation-derived antigens and will identify new antigens for T-cells mediated cancer immunotherapy that would directly influence cancer treatment strategies (e.g. cancer vaccines) by providing new target antigens for broader coverage on their own and in combination with existing therapeutic approaches. By combining immunology, computational biology, and clinical insights, this project addresses unmet needs in cancer treatment, fostering innovation in both research methodologies and therapeutic strategies.

This project stands on contributions from the expertise of immunology, clinical cancer research, and richness of computational analysis, thus creating a highly interdisciplinary approach to uncover the impact of splicing-derived antigens for cancer immunotherapy. The PhD candidate will be performing genomic analysis together with the Gronbeck group, utilizing clinical expertise, to identify patients with specific splice mutations, and will be working in the Supek group to establish a computational pipeline enabling identification of alternative transcripts, which would be experimentally validated using established tools of T-cell analysis.

Clinical insight and biological material: The clinical partner has long-standing expertise in the treatment and management of patients with hematological cancers. For this project, the partner will generate the specific information needed for selecting patient samples with splice mutations and the biological material will be accessed from established biobanks. Furthermore, critical information related to treatment modalities and disease outcomes will be shared to correlate with the T-cell data.

Computational analysis and antigen prediction tools: Computational methods enable a high-throughput, scalable approach to understanding splicing alterations, generating an actionable list of potential targets for further immunological validation. Supek group has extensive expertise in machine learning and evaluating genomic alterations. These will be leveraged to develop algorithms to detect cancer-specific alternatively spliced transcripts, particularly those potentially translated into antigens.

Immunology and T-cell detection technologies: Immunology drives the hypothesis that alternative splicing events in cancer generate antigens capable of eliciting a cytotoxic T-cell response, offering therapeutic avenues. The SKS group has developed high-throughput T-cell analysis technologies and has long-standing experience in identifying cancer antigens and their assessment in different therapeutic settings. Combining biological samples, identified splice-antigens using computational approaches, and immunological tools to assess the biological relevance would lead to identifying previously overlooked splicing-derived antigens, broadening the repertoire of immunotherapy targets. The project's outputs could redefine antigen discovery pipelines and improve cancer immunotherapy precision and effectiveness.